1. A genetic polymorphism in the Drosophila insulin receptor suggests adaptation to climate variation across continents Annalise Paaby a, Mark Blacket b, Ary Hoffmann b and Paul Schmidt a a) University of Pennsylvania b) University of Melbourne Natural populations of Drosophila melanogaster show variation in many life history traits. High latitude populations live longer and tolerate cold stress better than low latitude populations, but low latitude populations have higher rates of fecundity. These phenotypic patterns may reflect classical life history tradeoffs and represent adaptation to climate variation across geography. Fly populations vary in life history across latitude. North America: 9 populations Australia: 17 populations N-Q H Q Q Q H H - - Y Q H - H H - - - - - - - Q Q H L Q R R Q Q -C Q H Q Q Q H H H H Y Q H - H H - - - - - - - Q Q H H Q R R Q Q 248 Q H Q Q Q H H H H Y Q H - - H Q H H - - - - Q Q H H Q R R Q Q Q H Q Q Q H H H Y Q H H - - - - - - - - - - Q Q H L Q R R Q Q Q H Q Q Q H H H Y Q H H Q H H - - - - - - - Q Q H L Q R R Q Q Q H Q Q Q H H H Y Q H H Q H H Q H H - - - - Q Q H L Q R R Q Q 254 Q H Q Q Q H H H Y Q H H Q H H Q H H Q H H H Q Q H L Q R R Q Q The indel shows very similar patterns between the continents. The two most common alleles show inverse clines in both North America (248 p<0.01, 254 p=0.01) and Australia (248 p<0.01, 254 p<0.01). QUESTION: Can we identify adaptive genic elements by surveying allelic variation across environment? We observed an amino acid indel polymorphism in the first exon at InR that disrupts glutamine-histidine repeats. We saw a total of seven indel alleles in our sequence dataset. The most common alleles show a trend in frequency across latitude, but these samples only span three latitudinal points across North America. QUESTION: Do genes in the insulin signaling pathway vary significantly in the wild? We determined 52 InR sequences (7.3 kb) from populations at three regions across the latitudinal gradient of the U.S. east coast. PolymorphismDivergenceSelection est =0.0049MK test: p=0.03 Folded S: est =4.09 (CI=1.85-11.82)‏ indels=15Polym: S=88 NS=15 Folded NS: est =0.42 (CI=- 2.18-2.63)‏ SNPs=177Fixed: S=101 NS=37 Unfolded S: est =1.98 (CI=0.24-18.71) We saw evidence of protein evolution (MK test) and positive selection ( est ) at InR. These results contrast with evidence for neutral evolution at chico (data not shown), the InR substrate, which has a similar mutant phenotype. Additional sampling shows that the indel polymorphism is clinal. The indel polymorphism appears functional and adaptive. The insulin signaling pathway is well conserved in metazoans and regulates traits important for life history. Reduction in insulin signaling by mutation genetics extends lifespan, increases stress tolerance and decreases reproductive success in flies, worms and mice. These pleiotropic phenotypes mirror the natural history tradeoffs observed in the wild. We hypothesize that genes in the insulin signaling pathway may be targets of natural selection and components in a suite of genes and traits that respond adaptively to climate variation. Insulin signaling affects life history phenotypes. The Insulin-like Receptor shows evidence of adaptive evolution. An amino acid indel shows a pattern across latitude. We screened the indel polymorphism at higher sample size across North America and Australia. Introduction Results ACKNOWLEDGEMENTS We thank J. Plotkin for help in sequence analysis, and W. Eanes and T. Morgan for population samples. The indel shows independence from other sites. QUESTION: Is the clinal pattern across geography due to linkage with another site? InR is near the In(3R)Payne inversion on the third chromosome, previously shown to vary clinally and experience selection. However, the indel cline persists in lines carrying only standard and only inverted chromosomes, indicating independence (data not shown). Evaluation of natural genetic variation at candidate genes can identify regions of genic function. Nonrandom geographical distribution and an association with predictable variations in life history phenotypes suggest that the InR indel polymorphism is functionally important and contributes to observed differences in Drosophila life history. InR may contribute to Drosophila life history evolution. Conclusion QUESTION: Does the indel polymorphism affect phenotype? We tested lines carrying the 248 allele against lines carrying the 254 allele (in a randomized genetic background) for stress tolerance and fecundity. The 248 lines better tolerated cold stress (p=0.0305)) and oxidative stress (p=0.0007). The 254 lines laid more eggs (p=0.0030). These results accord with phenotypic effects of laboratory-induced mutations, which show higher stress tolerance and lower fecundity when insulin signaling is reduced. These results are also consistent with observations of higher stress tolerance at high latitudes, where the 248 allele is common, and higher fecundity at low latitudes, where the 254 allele is common. Of the 177 observed SNPs at InR, 11 are in linkage disequilibrium with the indel. Nine of these are close neighbors; the other two are synonymous sites. Decay of LD is observed both 5' and 3' of the indel polymorphism.